The longterm goals ofthe project are to identify the full array of efflux pumps of Staphylococcus aureus that contribute to multiple antimicrobial resistance and to elucidate the determinants of their expression, their role in microbial physiology and their effect on bacterial response to antimicrobials in infection. The work will focus on genetic analysis of regulatory elements and on bacterial fitness and response to antimicrobials in a subcutaneous abscess model, collaborating with other project groups to assess the efficacy of novel antimicrobial compounds in abscesses and the extent to which efflux pumps affect that efficacy. There are four specific aims: 1) assess the effects of NorB and Tet38 multidrug efflux pumps on response to antimicrobials in an abscess model; 2) assess the role ofthe newly identified NorD multidrug efflux pump in bacterial fitness and response to antimicrobials in an abscess model; 3) evaluate the regulation of expression of abcA encoding an ABC family efflux pump that confers resistance to p-lactams; and 4) determine the efficacy of targocil and other lead compounds identified in the program project in a mouse model of subcutaneous abscesses formed by community MRSA and characterize the occurrence and fitness costs of mutants resistant these compounds and the role of combination therapy with p-lactams. The work will utilize genetic manipulation and allelic exchange in S. aureus, measurements of gene expression with RT-PCR, and an established murine abscess model utilizing a genomically defined community strain of methicillin-resistant S. aureus. The overall goal ofthe program project is to take a well-integrated, multi-disciplinary approach to understanding antibiotic resistance development and transmission, and to integrate that effort with the search for compounds that compromise resistant pathogens, including methicillin-resistant S. aureus (MRSA), by inhibiting novel targets and pathways. This project will add to understanding of resistance mechanisms related to multidrug efflux pumps and provide strains for testing the effect of such pumps on novel compounds active against new targets and pathways. It will also utilize a mammalian model of a common MRSA infection to test compound activity and fitness of resistant mutants in vivo. RELEVANCE (See instructions): Multidrug resistance in S. aureus is an increasing clinical and public health problem that requires additional understanding of its mechanisms of development and spread and establishment of novel targets that may be exploited to develop new effective therapies.